Influenza A (fluA) infection causes annual substantial morbidity and mortality worldwide, particularly for infants, the elderly, and the immuncompromised. FluA mainly replicates in the respiratory tract, with virulent strains also disseminating to other tissues, such as the central nervous system. A growing concern is the recent spread of virulent avian influenza viruses, such as H5N1, in Asia, which could substantially increase the risk of a human pandemic. Sporadic cases of avian-to-human transmission, with a high mortaility rate have recently occurred. Because of its ability to readily undergo genetic reassortment and be generated by recombinant DNA, and to be rapidly spread by exposure to respiratory secretions, FluA is also a potential biothreat agent. Current vaccines, such as the parenterally administered trivalent inactivated vaccine (TIV) or live attenuated vaccine (LIV) given intranasally, induce minimal or modest levels of cytolytic T-lymphocyte (CTL) activity, and provide protection mainly by the production of CD4 T-cell dependent neutralizing antibodies. The efficacy of these vaccines is highly dependent on close matching of the hemagglutinin (HA) and neuraminidase (NA) surface proteins of the vaccine with currently circulating virus. Should neutralizing antibody fail to prevent infection of the respiratory tract, subsequent clearance of viral infection is mainly dependent on T cells, particularly CTL. Juvaris BioTherapeutics is developing and characterizing a unique adjuvant that is particularly promising for vaccines that induce both high levels of antibody and T-cell immunity, including CTL. The adjuvant is based on a complex of lipid carrier and non-coding DNA (CLDC), and is effective via multiple routes, including parenteral, respiratory, and oral. Inclusion of protein antigens with CLDC results in an extremely robust humoral and CD4 and CDS T-cell response to model antigens, such as ovalbumin or moth cytochrome c, as well as antigens from pathogens that target the respiratory tract, such as Yersinia pestis and Mycobacterium tuberculosis. Certain vaccination routes, such as intraperitoneal and oral administration results in particularly striking levels of CTL in the lung. The specific goals of this proposal are to optimize the CLDC- vaccine system for influenza A-specific T-cell responses in vivo, particularly in the lung. This optimal formulation will be used to evaluate protection against intranasal challenges with mouse attenuated influenza A strains (HKx31 and PR/8/34) as well as highly virulent recent isolate strains of avian H5N1 virus. A major focus will be in comparing vaccine efficacy based solely on T-cell immunity to internal virion proteins with that afforded by these responses in combination with antibody to external proteins. If successful, Phase II will examine potential manufacturing methods for the vaccine and large animal efficacy studies. [unreadable] [unreadable]